ICANCER RESEARCH56, 3404-3408, August 1, 19961 Advances in Brief Antiangiogenic Treatment with Linomide as Chemoprevention for Prostate, Seminal Vesicle, and Breast Carcinogenesis in Rodents1 Ingrid B. J. K. Joseph, Jasminka Vukanovic, and John T. Isaacs2 Johns Hopkins Oncology Center [A B. J. K. J., J. 1'.1.] and Department of Urology If. 1'. 1.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and Vanderbilt University Hospital If. V.], Nashville. Tennessee 37232-2358 Abstract There are two distinct phases during prostatic carcinogenesis with regard to tumor blood vessel development. During the first or prevas cular phase, which may persist for years, cells that have undergone some but not all of the transformation steps undergo a limited amount of net growth, producing premalignant prostatic intraepithelial neo plastic (PIN) lesions. Most of these PIN lesions do not continue net growth and do not progress to produce histologically detectable cancer. Even the PIN lesions that do progress to cancer remain of limited virulence unless they undergo conversion to the second or angiogenic phase. Once this angiogenic phase is reached, new blood vessel devel opment is greatly enhanced within the cancer. It is this enhanced tumor angiogenesis which allows these cancers both to grow continuously and to metastasize. Thus, inhibition of angiogenesis should be an effective chemopreventive approach for prostatic carcinogenesis. Linomide is a low molecular weight, water-soluble agent with excellent p.o. absorp tion and bioavailability. We have previously demonstrated that daily p.o. treatment with Linomide has antiangiogenic abilities against a series of rat and human prostatic cancer xenografts growing in vivo. In the present studies, we have demonstrated using Matrigel in in vivo angiogenesis assays that daily p.o. Linomide at 25 mg/kg/day inhibits angiogenesis induced by tumor necrosis factor a, acidic fThroblast growth factor, basic fibroblast growth factor, and vascular endothelial growth factor. Using an N-methylnitrosourea initiation-androgen promotion model, Linomide was given p.o. at a daily dose as high as 25 mg/kg/day for at least 1 year without major toxicity while inhibiting the development of seminal vesicle/prostate cancers in male rats by > 50% . Dose-response analysis demonstrated that a Linomide blood level of 50—100gLM is optimal for such chemoprevention. In addition, Linomide treatment at a dose of 25 mg/kg/day was able to inhibit by @6O% the incidence of N-methylnitrosourea and —50%of 7,12- dimethyl-benz(a)anthracine-induced mammary carcinogenesis in fe male rats. Introduction Over the last 50 years, the superficially benign nature of androgen ablation therapy has tended to disguise the fact that metastatic pros tatic cancer is still a fatal disease for which no therapy is available that is curative (1). New approaches are thus urgently needed. One ap proach is to develop modalities to prevent the initial development of clinically manifested prostatic cancer and/or its progression to a metastatic state by inhibiting one of the steps in prostatic carcinogen esis. Cells that have undergone some but not all of the transformation steps can undergo a limited amount of growth, producing morpholog ically detectable premalignant prostatic intraepithelial neoplastic le sions within the prostate (2—5).Not all of these premalignant lesions Received 4/29/96; accepted 5/29/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported by NIH Grant CA62482. 2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center, 422 North Bond Street, Baltimore, MD 21231-1001. progressed to produce histologically detectable prostatic cancer. In addition, once they progressed to the histologically detectable state, most of these prostate cancers remain clinically latent; i.e. , they do not progress to produce clinical symptoms during the lifetime of their host (2, 6). One explanation for this latency has been proposed based on the role of the development of new blood vessels (i.e., angiogenic) in prostatic carcinogenesis (7—10). Folkman et a!. (11) have demonstrated that induction of angiogen esis is a critical step in carcinogenesis involving the conversion of hyperplastic lesions with low tumorigenic ability into cancerous le sions which can produce continuously growing tumors. These studies demonstrated that the angiogenic ability appears first in a subset of hyperplastic lesions before onset of tumor, demonstrating that hyper plasia per se does not require angiogenesis. Furusoto and colleagues (9, 10) demonstrated that the majority of latent prostatic cancers detectable in autopsy material from men who died with no clinical indication of prostatic cancer had very low blood capillary density ratios compared to prostatic cancers which produced clinical symp toms and metastasized. It has been demonstrated that the intensity of angiogenesis within a variety of human cancers, including prostatic, can predict the metastatic ability of the cancer (7, 8). Coupling these observations with the demonstration that the growth of primary tu mors beyond 2—3mm3 is critically dependent on the induction of tumor angiogenesis (12) suggests that inhibition of angiogenesis should be a highly effective method for chemoprevention of prostatic cancer. Linomide, a quinoline-3-carboxamide, is a p.o. active agent which we have demonstrated previously to inhibit tumor angiogenesis and thus blood flow in a variety of rat prostatic cancers independent of their growth rate, androgen sensitivity, or metastatic ability (13—16). Likewise, it inhibits angiogenesis in human prostatic cancer xe nografts when grown in SCID mice (17). Because of its antiangio gemc affects, Linomide induces hypoxia-activated programmed death of prostatic cancer cells, thus inhibiting their net growth and meta static ability (15—17). The mechanism for Linomide's antiangiogenic effect involves its ability to inhibit the motility and invasiveness of both endotheial cells and macrophages (14, 15). Tumor-associated macrophages have been implicated in both positive and negative effects on tumor growth (18). One mechanism for positive effects of macrophages is through their ability to stimulate tumor vasculariza tion secondary to their secretion of various angiogenic factors, includ ing fibroblast growth factor, VEGF,3 and TNF-a (19). Linomide treatment not only decreases the number of tumor-associated macro phages within prostatic cancers but also decreases the ability of macrophages to synthesize and secrete the angiogenic factor TNF-a 3 The abbreviations used are: VEGF, vascular endotheial growth factor; TNF-a, tumor necrosis factor a; bFGF, basic fibroblast growth factor; aFGF, acidic fibroblast growth factor, OM, Osborne-Mendel; MNU, N-methylnitrosourea; TP, testosterone propionate; DHT, 5a-dihydrotestosterone; DHT-P, DHT propionate; DMBA, 7,12-dimethylbenz(a)- anthracine. 3404